title: Suppression of cardiac troponin T induces reduction of contractility and structural disorganization in chicken cardiomyocytes.
abstract: We herein examine the effect of cardiac troponin T (CTnT) suppression in cultured chicken cardiomyocytes derived from embryonic cardiac ventricular muscle. TnT is an important protein participating in regulation of striated muscle contraction, but it is not clear whether TnT contributes to the formation of sarcomere structure in myofibrils. Double-stranded RNA homologous to the nucleotide sequence of CTnT (CTnT-siRNA) was introduced into cultured muscle cells two days after plating. Transfection efficiency was above 80%. Immunoblot analyses suggested that the expression of CTnT progressively falls for the three consecutive days after transfection, but partly reappears on the fourth day. Maximum suppression occurs three days after transfection, with almost invisible CTnT protein on immunoblots in all the examined conditions: 0.5-2 nmol CTnT-siRNA towards 1-3 x 10(6) cells. The suppression was specific to CTnT, and the other myofibrillar proteins such as myosin, connectin/titin, tropomyosin, alpha-actinin, and troponin I were all present in transfected cells. The following functional and morphological changes were detected in CTnT-suppressed cells. The population of beating cells decreased significantly after transfection, when compared to control cells. A part of CTnT-suppressed cells showed two non-overlapping types of morphological changes: 1) myofibrils presenting unusually long Z-Z intervals; 2) myofibrils with irregular small striations in cells not connected at their adhesion interfaces of a jagged-appearance. Thus, our results reveal that CTnT is important for stable beating in cultured ventricular muscle cells, and also to some extent, for maintaining myofibrillar structure and cell-to-cell adhesion.
authors.affiliation: Department of Environmental Biology, Kumamoto Gakuen University, Oe, Kumamoto, Japan.
authors.forename: Naoji
authors.initials: N
authors.lastname: Toyota
authors.forename: Hiromi
authors.initials: H
authors.lastname: Takano-Ohmuro
authors.forename: Lucia S
authors.initials: LS
authors.lastname: Yoshida
authors.forename: Masatake
authors.initials: M
authors.lastname: Araki
authors.forename: Kumiko
authors.initials: K
authors.lastname: Yoshinobu
authors.forename: Fumie
authors.initials: F
authors.lastname: Suzuki-Toyota
mesh_terms: D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion / Q000235:genetics; D002478:Cells, Cultured; D002645:Chickens; D005786:Gene Expression Regulation; D009200:Myocardial Contraction / Q000235:genetics*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012518:Sarcomeres / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013489:Suppression, Genetic; D014162:Transfection; D020107:Troponin T / Q000172:deficiency* / Q000235:genetics
chemical_list: D034741:RNA, Small Interfering; D020107:Troponin T